DNA ligases are divalent metal ion dependent enzymes that utilize ATP or NAD+ to catalyze phosphodiester bond formation between adjacent polynucleotide termini possessing a 3′-hydroxyl and a 5′-phosphate (Tomkinson A E, PNAS, 2006). DNA ligases are essential enzymes for DNA replication and repair and are universally found in eukaryotes, bacteria, archaea and many viruses. Depending on their origin of species, natural occurring DNA ligases may have many unique properties, for example, substrate specificity, sequence and domain organization, optimal reaction condition such as pH, temperature and salt tolerance.
All known DNA ligases perform the catalysis via a common pathway which involves three nucleotidyl transfer reactions (see, Lehman I R. et al, Science, 1974; and Lindahl T et al, Annu Rev Biochem, 1992; both of which are herein incorporated by reference in their entireties, particularly for the steps for ligation). In the case of ATP-dependent DNA ligases, the first step (step 1) involves the attack on the α-phosphate of ATP by ligase, which results in release of pyrophosphate and formation of a ligase-AMP intermediate. AMP is linked covalently to the amino group of a lysine residue within a conserved sequence motif. In the second step (step 2), the AMP nucleotide is transferred to the 5′-phosphate-terminated DNA strand to form a 5′-App-DNA intermediate. In the third and final step (step 3), attack by the 3′-OH strand on the 5′-App-DNA joins the two polynucleotides and liberates AMP.
T4 DNA ligase was one of the first DNA ligases isolated (Weiss B, et al, PNAS, 1967), and the enzyme has since been widely used as a tool in molecular biology applications as well as molecular diagnostics, including cloning, sequencing, and gene synthesis etc. T4 DNA ligase readily accepts both nicked double-stranded DNA and double-stranded breaks with complementary base pairing. Furthermore, it is unique in its ability to join DNA fragments with blunt ends or single-base overhangs, even in the absence of a ligation enhancer, such as polyethylene glycol (PEG) or other small molecules (see, Sogaramella V et al, JMB, 1972; U.S. Pat. No. 8,697,408). For this reason, T4 DNA ligase is routinely used in many in vitro applications such as the library preparation workflow for high-throughput sequencing or next-generation sequencing (NGS sequencing).
It is common in many molecular biology applications to ligate double-stranded oligonucleotide adaptors to a library of double-stranded DNA fragments. For example, adapter ligation is an important step in the library preparation workflow of NGS sequencing. Attachment of double-stranded oligonucleotides with designed and known sequences to a library of DNA fragments with unknown sequences facilitates downstream manipulations, such as PCR amplification or primer extension. Efficient and complete ligation step ensures the success of the library preparation, and can reduce the number of cycles required for the PCR amplification of the library, which helps to reduce the necessary amount of starting material and minimize the bias in the resulting sequencing data. For these reasons, there is a need for improvement of the efficiency and completeness of the ligation reaction for many applications.